Recent advances in plant genetic engineering have opened new doors to engineer plants having improved characteristics or traits, such as, resistance to plant diseases, insect resistance, herbicidal resistance, enhanced stability or shelf-life of the ultimate consumer product obtained from the plants and improvement of the nutritional quality of the edible portions of the plant. Thus, a desired gene (or genes) from a source different than the plant, but engineered to impart different or improved characteristics or qualities, can be incorporated into the plant's genome. This new gene (or genes) can then be expressed in the plant cell to exhibit the desired new trait or characteristic.
It is important that the proper regulatory signals be present and be in the proper location with respect to the gene in order to obtain expression of the newly inserted gene in the plant cell. These regulatory signals include a promoter region, a 5′ non-translated leader sequence and a 3′ transcription termination/polyadenylation sequence.
A promoter is a short DNA sequence, usually upstream (5′) to the relevant coding sequence, to which RNA polymerase binds before initiating transcription. This binding aligns the RNA polymerase so that transcription will initiate at a specific site. The nucleotide sequence of the promoter determines the nature of the enzyme that attaches to it and the rate of RNA synthesis. The RNA is processed to produce messenger RNA (mRNA) which serves as a template for translation of the RNA sequence into the amino acid sequence of the encoded polypeptide. The 5′ non-translated leader sequence is a region of the mRNA upstream of the coding region that may play a role in initiation and translation of the mRNA. The 3′ transcription termination/polyadenylation signal is a non-translated region downstream of the coding region that functions in the plant cells to cause termination of the RNA and the addition of polyadenylate nucleotides to the 3′ end.
It has been shown that certain promoters are able to direct RNA synthesis at a higher rate than others. These are called “strong promoters”. Certain other promoters have been shown to direct RNA production at higher levels only in particular types of cells or tissues and are often referred to as “tissue specific promoters”. Many seed storage protein genes' promoters have been well characterized and widely used, such as the phaseolin gene promoter of Phaseolus vulgaris, the helianthinin gene of sunflower, the β-conglycinin gene of soybean (Chen et al., Dev. Genet. 10:112-122 (1989)), the napin gene promoter of Brassica napus (Ellerstrom et al., Plant Mol. Biol. 32:1019-1027 (1996)), the oleosin gene promoters of Brassica and Arabidopsis (Keddie et al., Plant Mol. Biol. 24:327-340 (1994); Li, Texas A&M Ph.D. dissertation, pp.107-128 (1997); Plant et al., Plant Mol. Biol. 25:193-205 (1994)). Another class of tissue specific promoters is described in U.S. Pat. No. 5,589,583, issued to Klee et al. on Dec. 31, 1996; these plant promoters are capable of conferring high levels of transcription of chimeric genes in meristematic tissues and/or rapidly dividing cells. “Inducible promoters” direct RNA production in response to certain environmental factors, such as heat shock, light, hormones, ion concentrations etc. (Espartero et al., Plant Mol. Biol. 25:217-227 (1994); Gomez-Gomez and Carrasco, Plant Physiol. 117:397-405 (1998); Holtorf et al., Plant Mol. Biol. 29:637-646 (1995); MacDowell et al., Plant Physiol. 111:699-711 (1996); Mathur et al., Biochem. Biophys. Acta 1137:338-348 (1992); Mett et al., Transgenic Res. 5:105-113 (1996); Schoffl et al., Mol. Gen. Genet. 217:246-253 (1989); Ulmasov et al., Plant Physiol. 108:919-927 (1995)).
Since the patterns of expression of a chimeric gene (or genes) introduced into a plant are controlled using promoters, there is an ongoing interest in the isolation and identification of novel promoters which are capable of controlling expression of a chimeric gene or (genes). Promoters that drive expression only in the developing seeds are of particular interest. Another desirable feature of a promoter would be an expression pattern that occurs very late in development in the developing seed or at seed maturity.